Isolation and characterization of a fluorene-degrading bacterium: identification of ring oxidation and ring fission products.

An Arthrobacter sp. strain, F101, able to use fluorene as the sole source of carbon and energy, was isolated from sludge from an oil refinery wastewater treatment plant. During growth in the presence of fluorene, four major metabolites were detected and isolated by thin-layer chromatography and high-performance liquid chromatography. 9-Fluorenol, 9H-fluoren-9-one, and 3,4-dihydrocoumarin were identified by UV spectra, mass spectrometry, and 300-MHz proton nuclear magnetic resonance. The fourth metabolite has been characterized, but precise identification was not possible. Since strain F101 is not able to grow with fluorenone, two different pathways of fluorene biodegradation are suggested: one supports cell growth and produces 3,4-dihydrocoumarin as an intermediate and probably the unidentified metabolite, and the other produces 9-fluorenol and 9H-fluoren-9-one and appears to be a dead-end route.     

Isolation and characterization of a fluorene-degrading bacterium: identification of ring oxidation and ring fission products.

An Arthrobacter sp. strain, F101, able to use fluorene as the sole source of carbon and energy, was isolated from sludge from an oil refinery wastewater treatment plant. During growth in the presence of fluorene, four major metabolites were detected and isolated by thin-layer chromatography and high-performance liquid chromatography. 9-Fluorenol, 9H-fluoren-9-one, and 3,4-dihydrocoumarin were identified by UV spectra, mass spectrometry, and 300-MHz proton nuclear magnetic resonance. The fourth metabolite has been characterized, but precise identification was not possible. Since strain F101 is not able to grow with fluorenone, two different pathways of fluorene biodegradation are suggested: one supports cell growth and produces 3,4-dihydrocoumarin as an intermediate and probably the unidentified metabolite, and the other produces 9-fluorenol and 9H-fluoren-9-one and appears to be a dead-end route.     

Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one.

Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

Microbial degradation of dibenzofuran, fluorene, and dibenzo-p-dioxin by Staphylococcus auriculans DBF63.

Staphylococcus auriculans DBF63, which can grow on dibenzofuran (DBF) or fluorene (FN) as the sole source of carbon and energy, was isolated. Salicylic acid and gentisic acid accumulated in the culture broth of this strain when DBF was supplied as a growth substrate. Also, the formation of 9-fluorenol, 9-fluorenone, 4-hydroxy-9-fluorenone, and 1-hydroxy-9-fluorenone was demonstrated, and accumulation of 1,1a-dihydroxy-1-hydro-9-fluorenone was observed when this strain grew on FN. On the basis of these results, the degradation pathways of DBF and FN were proposed. The analogous oxidation products of dibenzo-p-dioxin were obtained by incubation with DBF-grown S. auriculans DBF63 cells.     

Biotransformation of fluorene by the fungus Cunninghamella elegans.

The metabolism of fluorene, a tricyclic aromatic hydrocarbon, by Cunninghamella elegans ATCC 36112 was investigated. Approximately 69% of the [9-14C]fluorene added to cultures was metabolized within 120 h. The major ethyl acetate-soluble metabolites were 9-fluorenone (62%), 9-fluorenol, and 2-hydroxy-9-fluorenone (together, 7.0%). Similarly to bacteria, C. elegans oxidized fluorene at the C-9 position of the five-member ring to form an alcohol and the corresponding ketone. In addition, C. elegans produced the novel metabolite 2-hydroxy-9-fluorenone.     

Characterization of the upper pathway genes for fluorene metabolism in Terrabacter sp. strain DBF63

Genes involved in the degradation of fluorene to phthalate were characterized in the fluorene degrader Terrabacter sp. strain DBF63. The initial attack on both fluorene and 9-fluorenone was catalyzed by DbfA to yield 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone, respectively. The FlnB protein exhibited activities against both 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone to produce 9-fluorenone and 2'-carboxy-2,3-dihydroxybiphenyl, respectively. FlnD is a heteromeric protein encoded by flnD1 and ORF16, being a member of the class III two-subunit extradiol dioxygenase. FlnE was identified as a serine hydrolase for the meta-cleavage products that yield phthalate.     

Fluoranthene degradation in Pseudomonas alcaligenes PA-10

Pseudomonas alcaligenes strain PA-10 degrades thefour-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysisof the growth medium identified four intermediates, 9-fluorenone-1-carboxylicacid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formedduring fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acidand 9-hydroxy-1-fluorene-carboxylic acid resulted inincreases in fluoranthene removal, while pre-exposure to9-fluorenone and 9-fluorenol resulted in a decrease influoranthene degradation. The rate of indole transformation was similarly affected by pre-exposureto these metabolic intermediates, indicating a link between fluoranthenedegradation and indigo formation in this strain.     

Regiospecific and stereoselective hydroxylation of 1-indanone and 2-indanone by naphthalene dioxygenase and toluene dioxygenase.

The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterial strains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purified enzyme components. Pseudomonas sp. strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixture of 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%). The (R)-3-hydroxy-1-indanone was formed in 62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic. The same cells also formed 2-hydroxy-1-indanone from 2-indanone. Purified NDO components oxidized 1-indanone and 2-indanone to the same products produced by strain 9816/11. P. putida F39/D cells, expressing TDO, oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone efficiently. Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90% ee (R:S, 5:95) and failed to oxidize 1-indanone. Oxidation of 1- and 2-indanone in the presence of [18O]oxygen indicated that the hydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation) rather than by the dioxygenation of enol tautomers of the ketone substrates. As alternatives to chemical synthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-1-indanone as the major products from 1-indanone and 2-indanone, respectively.     

Bacterial degradation of aromatic compounds via angular dioxygenation

Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds. Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol. This "normal" type of dioxygenation is termed lateral dioxygenation. On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation. In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized. Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively. Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure. The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl. 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis. Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl. Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported. Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp. strain DBF63, and carboxylated DE-degradation pathway of P. pseudoalcaligenes strain POB310, have been investigated at the gene level. As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily. Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.     

Transformation of Substituted Fluorenes and Fluorene Analogs by Pseudomonas sp. Strain F274

Pseudomonas sp. strain F274, previously shown to catabolize fluorene via fluorenone and its angular dioxygenation, 2(prm1),3(prm1)-dihydroxy-2-carboxybiphenyl, phthalate, and protocatechuate, was examined for its ability to transform substituted fluorenes and S- and N-heterocyclic analogs. Halogen- and methyl-substituted fluorenes were metabolized to correspondingly substituted phthalates via attack on the unsubstituted ring. In the case of 1-methylfluorene, initial oxidation of the methyl group to carboxyl prevented all other transformations but 9-monooxygenation. This strain also oxidized the S-heteroatoms and benzylic methylenic groups of fluorene analogs. No angular dioxygenation of S- and N-heterocycles was observed.     

Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans

The preferred substrates in angular dioxygenation, monooxygenation, and lateral dioxygenation by dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas resinovorans strain CA10 are shown to be distinctly different. The preferred oxygenation reactions suggest that DFDO evolved from a polycyclic aromatic hydrocarbon dioxygenase and that its most preferred substrates were fluorene and 9-fluorenone. The angular dioxygenases involved in the degradation pathway of dibenzofuran (dioxin) and fluorene are closely related in function, while CARDO is a novel enzyme not only phylogenetically but also functionally.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469

Biodegradation of fluorene by Rhodococcus rhodochrous VKM B-2469 was investigated and optimized by adding non-ionic surfactants to the liquid media. The utilization of 1-1.5% Tween 60 or 1% Triton X100 allowed to solubilize 1 mM fluorene over 150 times more than in water medium (from 9-11 microM to above 1.5 mM at 28 degrees C). We observed that Tween 60 was useful to enhance the fluorene biodegradation rates further supporting R. rhodochrous VKM B-2469 growth as an additional carbon source and to decrease fluorene toxicity for bacterial cells whereas Triton X100 resulted to be toxic for this strain. An additional enzyme induction step before starting the bioconversion process and the increase of incubation temperature during fluorene bioconversion led to further improvements in rates of fluorene utilization and formation of its intermediates. In the optimized conditions 1 mM fluorene was degraded completely within 24h of incubation. Some intermediates in fluorene degradation built up during the process reaching maxima of 31% for 9-hydroxyfluorene, 2.1% for 9-fluorenone and 1.9% for 2-hydroxy-9-fluorenone (starting from 1 mM substrate). In the presence of Tween 60 the appearance and following conversion of 2-hydroxy-9-fluorenone was observed for R. rhodochrous VKM B-2469 revealing the existence of a new pathway of 9-fluorenone bioconversion.     

Polycyclic aromatic hydrocarbons degradation by Agrocybe sp. CU-43 and its fluorene transformation

Agrocybe sp. CU-43, a white-rot fungus isolated from Thailand, showed a high potential for degrading both low- and high-molecular weight polycyclic aromatic hydrocarbons. At 100 ppm fluorene was degraded by 99% within six days while at the same concentration 99 and 92% degradation of phenanthrene and anthracene, respectively, occurred in 21 days, and fluoranthene and pyrene were reduced by 80 and 75%, respectively, in 30 days. In a soil model, Agrocybe sp. CU-43 completely degraded 250 ppm fluorene at room temperature within four weeks. Laccase and manganese peroxidase activities, but not lignin peroxidase activity, were detected during the biodegradation of fluorene. Two of the metabolites from fluorene degradation by the fungus were identified via reversed-phase HPLC as 9-fluorenol and 9-fluorenone, the less toxic intermediates of fluorene. However, 9-fluorenol is not an end product for the degradation. These results suggest that fluorene degradation by Agrocybe sp. CU-43 may take place via the same pathway(s) employed by other ligninolytic and non-ligninolytic fungi. This is the first report of fluorene biodegradation by a fungus belonging to the genus Agrocybe.     

Oxidative transformation of 2-acetylaminofluorene by a chemical model for cytochrome P450: A water-insoluble porphyrin and tert-butyl hydroperoxide

Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical model was 2-nitro-9-fluorenone (NO(2)=FO), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF-OH), and then oxidized further to 2-acetylamino-9-fluorenone (AAF=O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO(2)F), and further oxidation yielded 2-nitro-9-fluorenol (NO(2)F-OH) as a minor product. These products, AAF-OH, AAF=O, NO(2)F, and NO(2)F-OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model, and the formation of NO(2)F=O was determined. These results showed that NO(2)F=O was the mutagen derived from AAF in the presence of the chemical model and was formed via oxidation of N-OH-AAF, NO(2)F, and NO(2)F-OH. These results may lead to a new metabolic pathway of AAF.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.     

Dioxygenolytic cleavage of aryl ether bonds: 1, 10-dihydro-1, 10-dihydroxyfluoren-9-one, a novel arene dihydrodiol as evidence for angular dioxygenation of dibenzofuran

Two dibenzofuran degrading bacteria, Brevibacterium strain DPO 1361 and strain DPO 220, were found to utilize fluorene as sole source of carbon and energy. Cells which were grown on dibenzofuran, transformed fluorene into a number of products. For five of the seven metabolites isolated, the structure could be established unequivocally. Accumulation of one metabolite, 1,10-dihydroxy-1,10-dihydrofluoren-9-one, indicated the presence of a novel type of dioxygenase, attacking polynuclear aromatic systems in the unusual angular position. Debenzofuran degradation is proposed to likewise proceed via initial angular dioxygenation. One aryl oxygen ether bond, which normally is extremely stable, is thus transformed to a hemiacetal. After spontaneous cleavage and subsequent rearomatization by dehydration, 2,2',3-trihydroxybiphenyl [3-(2-hydroxyphenyl)-catechol] thus results as the immediate product of the first enzymatic reaction in the degradation sequence.